The inclusion of a chaotropic agent, such as urea, in both starch and acrylamide gels has permitted electrophoretic separation in these gels of certain classes of proteins or nucleic acids which would not otherwise be separated. Since chaotropic agents in high concentrations prevent the formation of hydrogen bonds, protein complexes or aggregates, the structure of which is maintained by such bonds, are readily disassociated in the presence of these agents. Separation may thus be achieved, for example, by diluting the sample mixture in a urea solution and separating it electrophoretically in a gel which contains urea. As is well known, protein complexes or aggregates or nucleic acids are readily disassociated in urea at room temperature without undergoing a change in charge. Urea-containing gels are thus highly useful in isoelectric focussing, both alone and at the first stage of two-dimensional electrophoresis.
It is known that polyacrylamide gels, formed from acrylamide monomers with the inclusion of bisacrylamide (N,N'-methylenebis(acrylamide)) as a cross linking agent with urea included as a solubilizer, must be used soon after they are formed. If such gels are stored more than 24 hours at room temperature, their ability to separate proteins deteriorates significantly. While refrigeration lessens the rate of deterioration, it gives rise to another problem, urea crystallization Such crystallization occurs, for example, in gels with 9M urea at 4.degree. C. within a few hours.
The deterioration of the gels precludes precasting and storage for any appreciable period of time, and seriously detracts from the ease of using such gels. The need to prepare the gels within a few hours of their use not only limits the operator's efficiency, but also creates the possibility of variations from one gel to the next, introducing non-reproducibility, as well as increasing the likelihood of errors and lost time due to unusable data.
It has now been discovered that the problems of both gel deterioration and urea crystallization are eliminated by the use of diacrylyl compounds containing tertiary amide groups as cross linking agents. While the acrylyl moieties in the compounds provide the compatibility with the polyacrylamide chains which is characteristic of the bis-acrylamide cross linking agent, the tertiary structure of the nitrogens as opposed to their secondary structure in bis-acrylamide is unexpectedly found both to impart a long term stability to the gel and to function as an "anti-freezing" agent as well. The latter is particularly surprising in view of the ineffectiveness of certain additives known for their anti-freezing properties in other media.